Succinylcholine-Induced Acceleration and Suppression of Electrically Evoked Acetylcholine Release from Mouse Phrenic Nerve-Hemidiaphragm Muscle Preparation

Ikuko Kimura, Marl Okazaki, Teruko Uwano, Shinjiro Kobayashi and Masayasu Kimura

Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-01, Japan

Received April 24, 1991 Accepted August 20, 1991

ABSTRACT-The effects of a depolarizing neuromuscular blocker on electrically evoked acetylcholine (ACh) release were studied using a mouse phrenic nerve-dia phragm muscle preparation preloaded with [3H]-choline, and the changes in muscle tension were recorded simultaneously. Succinylcholine at a low concentration (1 ,uM) enhanced evoked [3H]-ACh release, which tended to follow the increase in peak am plitude of tetanic tension; whereas at high concentrations (10 and 30,uM), it simul taneously reduced both release and tension. Decamethonium even at 10 and 30,uM had little effect on [3H]-ACh release despite producing a significantly greater reduc tion in tension compared with succinylcholine. (+)-Tubocurarine (5 PM) prevented the enhancing effect of [3H]-ACh release induced by 1 uM, but not the decreasing effect induced by 10 uM succinylcholine. These results suggest that succinylcholine in duced acceleration at low concentrations due to a positive feedback mechanism through presynaptic nicotinic ACh receptors and the inhibition of ACh release at high concentration contributes in part to the neuromuscular blockade.

Presynaptic acetylcholine (ACh) receptors radioisotope method to count [3H]-efflux in modulate ACh release from motor nerve ter the absence of cholinesterase inhibitors. The minals (1, 2). Electrophysiological and mecha change in muscle tension was recorded simul nical measurements suggest that (+ )-tubocur taneously in the same preparation to clarify its arine may reduce the evoked release of ACh relation to ACh release. through blockade of a positive feedback mechanism mediated by presynaptic nicotinic MATERIALS AND METHODS ACh receptors (3, 4). The presynaptic mecha nism of neuromuscular blockade induced by Male ddY mice (7 -10 weeks old, 31 41 g) depolarizing blockers such as succinylcholine were decapitated and bled. Either the right or and decamethonium has not yet been eluci left phrenic nerve-diaphragm muscle was re dated in detail. moved and cut into strips about 10-mm wide The aim of the present study was to deter together with the attached rib segment. The mine whether succinylcholine has a direct rib end of the preparation was pinned to rub effect on ACh release from the motor nerve ber plates in a chamber, and the tendon was endings of mouse diaphragm using the tied with a silk thread and connected to an isometric transducer. The strip was suspended The absolute amount of spontaneous and in a 2-ml chamber in Krebs solution gassed electrically evoked output of ACh from the with a mixture of 95% 02 and 5% C02 at preparation was determined in part by 37°C. Krebs solution was composed of 113 radio immunoassay (8, 9) using ACh-anti mM NaC1, 4.7 mM KCI, 2.5 mM CaC12, 1.2 serum. The experimental protocol was the mM MgSO4, 25 mM NaHCO3, 1.2 mM same as that described above except for in KH2PO4, and 11.5 mM glucose. cubation with [3H]-choline. The preparation was treated with 10 mM methanesulfonyl Measurement of f 3H]-ACh in the perfusate fluoride, an irreversible acetylcholinesterase A radioisotope method was used to measure inhibitor, during incubation. The perfusate the electrically evoked release of ACh without was collected continuously on ice. Then 630,ul cholinesterase inhibitors (5 7). The above of perfusate was incubated for 17 hr with 100 ,ul preparation was incubated for 60 min in 2 ml of diluted antiserum (1:300) in Tris-HCl buff Krebs solution containing methyl [3H]-choline er (0.1 M, pH 7.4) containing 0.4% bovine (370 kBq). To facilitate the uptake of [3H] gamma-globulin, 0.05% diisopropylfluorophos choline into the acetylcholine pool in motor phate, 10 ,u1 of 10 mM acetic acid and 60 ,u1 of nerve terminals, the preparation was stimu tritiated ACh (4.8 pg, 5500 d.p.m.). The same lated for 40 min. For the next 20 min, the volume of Krebs solution was used as a blank preparation was allowed to rest. The prepara test. Antibody-bound ACh was separated tion was then washed with Krebs solution at a from free tritiated ACh by the ammonium sul rate of 2 ml/min for 60 min to remove the ex fate method and measured in a liquid scintilla cess [3H]-choline. The perfusion rate was then tion counter. The presynaptic effect of suc slowed to 1 mUmin. The phrenic nerve was cinylcholine cannot be determined by radioim stimulated four times. The 3-min stimulation munoassay because of the high cross-reactivity periods started at 8 (So), 32 (SI), 56 (S2) and of ACh-antiserum to succinylcholine. 80 (S3) min after the washout period. Trains of 40 pulses (50 Hz, 0.2 msec duration, for 0.8 Peak amplitude of tetanic tension sec) were applied every 10 sec for 3 min by The isometric contractions were measured supramaximal square pulses (1-10 V) through with a force displacement transducer (Nihon a pair of platinum electrodes (7). Samples Kohden, SB1T) and recorded (Linea Record were collected every min from 9 min before er, Graphtec, WR3701). The resting tension the 32 min point (S,). The samples were col was adjusted to 500 mg. The effects of drugs lected into scintillation vials. Six milliliters of on the peak amplitude of tetanic tension were scintillation fluid (ACS-II, Amersham) was represented as T2/T,, where T was an aver added to each one-min aliquot of perfusate. aged value of the 6th, 12th, and 18th peak Radioactivity of the samples was measured amplitudes of tetanus for a 3-min stimulation in a scintillation beta spectrometer (Beckman, period. Tl and T2 were produced by S1 and S2 LS 3801). The stimulation-induced increase in stimulation periods, respectively. [3H]-release was calculated by subtracting the mean of the basal release from the total re Statistics lease. Mean basal release was calculated from The unpaired t-test or one way analysis of six data before and after a stimulation period. variance was used. P < 0.05 was adopted as The tritium content of the tissue was meas the level of significance. ured after the perfusion experiment by placing the preparation into 2 ml of 0.4N NaOH and Drugs counting a 50-,ul aliquot. Methyl [3H]-choline chloride (555 GBq/ mmol), acetylmethyl-[3H]-choline chloride Radioimmunoassay for ACh in the perfusate (2.78 TBq/mmol) (Amersham, Buckingham shire, England), succinylcholine chloride 2 cantly cross-reacted with micromolar concen H20, (+ )-tubocurarine 3.5 H2O (Nacalai Tes trations of succinylcholine (8), the effects of que, Kyoto, Japan), decamethonium bromide, succinylcholine were investigated by [3H] gamma-globulin bovine Cohn fraction II, III efflux counting. (Sigma, St. Louis, MO, U.S.A.), acetylcholine Typical data showing the succinylcholine chloride (Daiichi Seiyaku, Tokyo, Japan), and induced effects on the evoked [3H] release are methanesulfonyl fluoride (Aldrich, Milwau shown in Fig. 1. The S2/S, ratio was 1.36 ± kee, WI, U.S.A.) were used. The rabbit ACh 0.15 at 1 uM succinylcholine (n = 4), dem antiserum (NGC-10 880902, sensitivity of 10 onstrating a significant enhancement of pg/tube) was a gift from Prof. K. Kawashima evoked ACh release. The ratio was 0.41 ± (Department of Pharmacology, Kyoritsu Col 0.06 at 10,uM succinylcholine (n = 4), indicat lege of Pharmacy, Japan). ing a significant inhibition of ACh release. The concentration-response curve for the RESULTS ratios of evoked ACh release (S2/S1)was com pared with that for the ratios of peak ampli The absolute value of resting release before tude of tetanic tension (T2/T1) (Fig. 2). Both S1 was 2182 ± 383 Bq/(g of tissue) min' for curves showed the following biphasic patterns: [3H]-efflux (n = 4). Electrically evoked [3H] Low concentrations of succinylcholine en ACh release over the level of spontaneous hanced and high concentrations reduced both output as determined by [3H]-efflux counting ACh release and tetanic tension. Succinylcho was compared with the amount of ACh deter line at 1 ,uM clearly enhanced the S2/S, ratios, mined by radioimmunoassay in Table 1. The and 0.3 ,uM slightly enhanced the T2/T, ratios. S2/S, ratio was not altered when the prepara Both ratios were reduced significantly by 10 tion was treated with 10 mM methanesulfonyl ,uM and 30 pM succinylcholine. fluoride. The concentration-response curves of deca Radioimmunoassay showed that the abso methonium are shown in Fig. 3. Deca lute value of resting release before S, was 3.34 methonium at concentrations up to 10 ,u M did ± 0.25 ng/(g of tissue) min-' (n = 4). The not have any significant effect on evoked ACh absolute amount of ACh release was constant, release despite the large decrease in the peak and the S2/S, value was not significantly dif amplitude of tetanic tension at 10,uM. Even ferent from 1.0. Since ACh-antiserum signifi at a high concentration (30 ,uM), deca

Table 1. Electrically evoked acetylcholine release from mouse phrenic nerve-diaphragm mus cle preparation determined by [3H]-efflux counting compared with that determined by radioim munoassay effect on evoked ACh release, reduced the en hancement of ACh release induced by 1 ,u M succinylcholine, but did not antagonize the in hibition induced by 10,uM succinylcholine.

DISCUSSION

Presynaptic effects of succinylcholine have been proposed in addition to its postsynaptic effects. Succinylcholine in the presence of acetylcholinesterase inhibitor reduces the num ber of ACh quanta released from frog nerve muscle preparation (10). Succinylcholine affects the motor nerve terminal in much low er doses than those which cause neuromuscu lar blockade on the soleus nerves and muscles of cats (11). The neuromuscular blockade caused by succinylcholine is dominated by the partial failure of rat phrenic nerve terminals to release ACh in stretched, partially depolarized muscles (12). All these suggestions are based on electrophysiological data. ACh release from mouse phrenic nerve hemidiaphragm preparations is measured directly in the presence of hemicholinium-3 to Fig. 1. Typical data showing the effects of succinyl prevent the re-uptake of [3H]-choline (5-7). choline (SuCh) on [3H]-acetylcholine (ACh) release In the present study, both released ACh and from phrenic nerve stimulated electrically in mouse muscle tension were measured simultaneously nerve-hemidiaphragm muscle preparation. Muscles in response to electrical stimulation of the were incubated for 1 hr with [3H]-choline (electrical sti mulation for 40 min and rest for 20 min) and then phrenic nerve without a cholinesterase inhibi washed out for 1 hr. [3H]-ACh release was elicited by tor and without hemicholinium-3, which im two stimulation periods (S,, S2; 50 Hz and trains of 40 pairs ACh synthesis and produces a frequency stimuli were applied every 10 sec for 3 min) which were dependent fall-off in ACh output (4). indicated by the open columns. The shaded column in Our present study using the measurement of dicates the presence of succinylcholine (1 uM, middle and 10 MM, lower). Data are each 1-min value during [3H]-ACh overflow indicated that the effect of the stimulation period or mean values for 3 min during succinylcholine is characterized by a two-phase the resting period. The horizontal axis indicates the pattern as follows: Succinylcholine enhanced time after the end of the washout period. ACh release and peak amplitude of tetanic tension at low concentrations, whereas it re duced them at high concentrations. Evoked methonium reduced evoked ACh release by ACh release was increased by a low, but 3 only 35% despite the 100% reduction in ten times higher concentration of succinylcholine sion. than that at which tetanic tension tended to be The effects of 1 and 10,uM succinylcholine increased. Increase in evoked ACh release on evoked ACh release in the presence and was not accompanied with an increase in ten absence of 5 ,uM (+ )-tubocurarine were com sion because evoked tetanic tension may have pared (Fig. 4). (+)-Tubocurarine (5,uM) already been the maximal value. Therefore, alone, completely inhibiting tension, had no the increase in tension induced by succinylcho Fig. 2. Concentration-response curves of succinylcholine (SuCh) for evoked [3H]-ACh release (closed sym bols) and peak amplitude of tetanic tension (open symbols) in mouse phrenic nerve-diaphragm muscle preparation. Succinylcholine was administered in various concentrations 10 min before S2. Stimulation was applied as described in Fig. 1. Each point represents the mean for relative ratios of ACh release (S2/S1) and tension (T2/T,), and vertical lines represent S.E.M. (n = 4-6). T1 or T2 is an averaged value of the 6th, 12th, and 18th peak amplitudes of tetanus produced by S, or S2 stimulation period, respectively. Significant differences from the control (without drug) were analyzed by Student's t-test. * * P < 0.01 and * P < 0.05.

Fig. 3. Concentration-response curves of decamethonium (C,,,) for evoked [3H]-ACh release (closed sym bols) and peak tetanic tension (open symbols). Decamethonium was administered in various concentrations 10 min before S2. Stimulation was applied as described in Fig. 2. Points and vertical lines are the same as in Fig. 2 (n = 4 6). Significant differences from the control (without drug) as determined by Student's t-test. **P<0 .01. Fig. 4. Concentration-response curves of succinylcholine (SuCh) for evoked [3H]-ACh (closed symbols) and peak tetanic tension (open symbols) with (straight lines) or without (+)-tubocurarine (d-TC, 5 uM) (dotted lines replotted from Fig. 2). Succinylcholine and (+)-tubocurarine were simultaneously administered 10 min before S2. Points and vertical lines are the same as in Fig. 2 (n = 4). Significant differences from the values with succinylcholine alone were determined by the unpaired t-test: ttP < 0.01 and tP < 0.05.

line may not be due to the increase in evoked ACh release by a positive feedback mecha ACh release. Evoked ACh release was de nism, the evidence for which has been pro creased by high concentrations of succinylcho vided by electrophysiological (14), pharmaco line, which were accompanied by simultaneous logical (15, 3, 4) and radioisotope methods (16 suppression of the peak amplitude of tetanic -18) . Succinylcholine at low concentration tension to the same extent. Electrophysiologi may promote the positive feedback mechanism cal data suggest that the cause of the neuro by presynaptic nicotinic ACh receptors, the muscular block by succinylcholine is a post enhancing effect of succinylcholine on evoked synaptic inhibitory effect, rather than a pre ACh release was completely inhibited by (+ ) synaptic one (13). However, our results sug tubocurarine. (+ )-Tubocurarine may suppress gest that the mechanism of neuromuscular it by reacting with presynaptic n-ACh recep blockade induced by succinylcholine is in part tors. No inhibitory effect of (+)-tubocurarine due to presynaptic suppression. on ACh release in the presence of acetylcholin The two depolarizing neuromuscular block esterase inhibitor has been demonstrated (19, ers were quite different in their presynaptic 20): the desensitization of presynaptic nicotinic actions. In contrast to succinylcholine, deca receptors may develop rapidly in the presence methonium hardly affected the motor nerve of high concentrations of agonists or cholin terminal. The neuromuscular block induced by esterase inhibitors (16). Presynaptic nicotinic decamethonium is, therefore, attributable to receptors are more sensitive to desensitization postsynaptic inhibition alone. Succinylcholine than postsynaptic ones (21). The inhibitory ac had a greater effect on the motor nerve ter tion of succinylcholine was not antagonized by minal than decamethonium. (+ )-tubocurarine; thus the high concentra Presynaptic nicotinic receptors facilitate tions of succinylcholine may have desensitized presynaptic nicotinic ACh receptors. synaptic M, muscarinic receptors in guinea pig In conclusion, succinylcholine-induced accel ileum. J. Pharmacol. Exp. Ther. 244, 1036-1039 eration of ACh release at low concentration is (1988) 10 Edwards, C. and Ikeda, K.: Effects of 2-PAM and due to positive-feedback mechanisms involving succinylcholine on neuromuscular transmission in presynaptic nicotinic ACh receptors and the the frog. J. Pharmacol. Exp. Ther. 138, 322-328 inhibition of ACh release at high concentra (1962) tion may contribute to the neuromuscular 11 Standaert, F.G. and Adams, J.E.: The actions of blockade. succinylcholine on the mammalian motor nerve terminal. J. Pharmacol. Exp. 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